The ultimate objective of these investigations is to characterize HIV particle structures. Typically, HIV particles adopt two distinct morphologies: immature, and proteolytically processed mature forms. The natural variability of immature and mature HIV virions has made it extremely difficult to study these particle forms. However, it is clear that the HIV Gag proteins constitute the major structural determinants of both viral forms. Specifically, the Gag precursor (PrGag) proteins direct the assembly of immature virions on cellular membranes, while the processed Gag proteins matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1, and p6 organize mature virus assemblies. To understand how HIV Gag proteins associate to form higher order structures, we recently developed methods for the in vitro analysis of Gag proteins assembled on lipid membranes. Significantly, our results are consistent with in vivo observations, and have provided new insights as to how PrGag assembly occurs. The investigations described in this proposal are designed to extend our promising preliminary results, and to test our predictions using virus particles produced from mammalian cells. The results we obtain will provide crucial insights into mechanisms of assembly and morphogenesis that should serve as a foundation to attack HIV at its viral core. Our specific aims are as follows: 1. Electron diffraction-image analysis of Gag proteins assembled on lipid membranes: We will employ our novel method of assembling histidine-tagged (his-tagged) HIV Gag proteins on membranes containing nickel-chelating lipids to obtain three-dimensional structures of membrane-bound Gag proteins and their interactions. 2. Validation of structural models in vivo: The validity of results obtained from in vitro studies will be tested using cellularly derived virus particles, which will be examined biophysically, biochemically, and genetically to assess the accuracy and usefulness of structural models.